Selection of optimal extraction and RT-PCR protocols for stool RNA detection of colorectal cancer associated immune genes

There is a growing interest in using fecal mRNA transcripts as biomarkers for non-invasive detection of colorectal cancer (CRC). The following study compares different RNA extraction and reverse transcription PCR (RT-PCR) methods for mRNA detection in stool and identifies a robust and sensitive protocol. A combination of the Stool total RNA purification kit (Norgen) and the Superscript III one-step RT-PCR kit (Invitrogen) provided high RNA purity and sensitive and consistent mRNA detection, making them well-suited candidates for large-scale studies. We tested the protocol by detecting the mRNA of several immune genes ( CXCL1 , IL8 , IL1B , IL6 , PTGS2 , and SPP1 ) in 22 CRCs, 24 adenomatous polyps, and 22 control stool samples. All these inflammatory markers, except for CXCL1 , showed a strong association with CRC. Cancer stool samples showed increased levels of IL1B , IL8 , and PTGS2 transcripts compared to polyp and control groups . Thus, this work supports the potential use of fecal mRNA as biomarkers for CRC detection.